Pretreatment kit for saliva and pretreatment method for saliva

ABSTRACT

A pretreatment kit and a pretreatment kit for saliva in identification and quantitative determination of  mutans  streptococci by immunochromatography utilizing an antigen-antibody reaction, which can remove aggregation caused by mucin and chain formation of  mutans  streptococci in saliva in a simple operation and can efficiently flow out a complex of a labeled antibody and  mutans  streptococci from a porous membrane retaining the labeled antibody, contains (A) a 0.01 to 10 mol/L aqueous solution of sodium hydroxide, (B) a 0.01 to 3 mol/L aqueous solution of tartaric acid and/or citric acid, and (C) a nonionic surface active agent and/or an amphoteric surface active agent, in which the component (C) is mixed with the components (A) and/or (B), or is provided separately, and at least one substance selected from the particular metallic salts is contained in at least one of the components (A), (B) and (C) in an amount of 5 to 25% by weight.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a pretreatment kit for saliva foridentification and quantitatively determining mutans streptococci, asone of cariogenic bacteria in human saliva, by immunochromatographyutilizing an antigen-antibody reaction, and a pretreatment method forsaliva using the pretreatment kit for saliva.

2. Description of Conventional Art

It has been known that there is close relation between the presence ofmutans streptococci and the generation of dental caries in a humanmouth, and therefore, the morbidity risk and the current condition ofmorbidity can be comprehended to provide benefits to quite a number ofpeople if the presence or absence and the amount of mutans streptococciin a human mouth can be conveniently examined.

An examination technique utilizing an antigen-antibody reaction inexamining has been conventionally carried out. For example, theimmunoenzymatic technique, which is a method of identification andquantitatively determining with coloring density using an enzyme,requires a special washing device and complicated and accurateoperations for handling an antibody and a sample, and also requires anincubator for carrying out an enzyme reaction. The fluorescent antibodytechnique, which is a method of specifically staining an antigen that isreacted with an antibody labeled with a fluorescent dye, is not commonlyemployed since a fluorescent microscope is necessary as a measurementdevice.

Accordingly, various techniques have been proposed for convenientlyutilizing an antigen-antibody reaction. Examples thereof include ameasurement technique utilizing chromatography (as shown, for example,in U.S. Pat. Nos. 5,591,645, 4,855,240, 4,435,504 and 4,980,298, andJapanese Patent Application Publication Nos. JP-A-61-145459 andJP-A-6-160388). The technique is excellent in simpleness because thepresence or absence and the amount of an antigen can be measured only bymixing a body fluid thus collected in a test solution containing anantigen to be identified and quantitatively determined, and theninstilling in an examination device. The technique is generally referredto as an immunochromatography technique, and the principles ofidentification and quantitative determination have been disclosed indetail (as shown, for example, in Se-Hwan Peak, Seung-Hwa Lee,Joung-Hawan Cho and Young-Sang Kim, “Development of rapid One-Stepimmunochromatographic assay, Methods”, vol. 22, p. 53 to 60 (2000)).

It seems that identification and quantitative determination of mutansstreptococci in the human mouth can be carried out by applying thetechnique, but it has not been put into practical use because of thefollowing problems. That is, it is necessary that a sample used for theimmunochromatography technique pass through a porous membrane by thecapillary phenomenon. However, because the major sample applied to theexamination of bacteria in the mouth, such as mutans streptococci, issaliva, a high viscosity substance present in saliva, which is referredto as mucin, clogs the pores of the porous membrane. Furthermore,because mucin has such a function that aggregates epithelial attachmentcells stripped off from oral mucosa, the pores of the porous membraneare clogged with these substances to inhibit transmission of mutansstreptococci.

In addition to mucin, there is another problem complicatingidentification and quantitative determination of mutans streptococci bythe immunochromatography technique. That is, the mutans streptococci tobe measured is a bacterium having a diameter of about 1 μm solely butoften forms a chain with 10 to 20 or more bacteria owing to the natureof streptococci, which may be a factor of inhibiting migration in theporous membrane. Furthermore, the mutans streptococci forms glucanhaving adherence from sucrose in foods and is often severely aggregated.The chain formation and aggregation of mutans streptococci causeclogging in the porous membrane and also reduce the surface area of thestreptococci to affect quantitative determination of the number ofantigens present on the surface of the mutans streptococci, whichreduces accuracy of the measurement.

In the immunochromatography technique, detection of an analytic objectis generally carried out by using two antibodies. The first antibody isretained in a porous membrane formed with glass fibers or the like onthe side where a sample is dropped, and the antibody is generallylabeled with latex particles, gold colloid particles or the like(hereinafter, sometimes referred to as a labeled antibody). In the casewhere the analytic object to be measured is present in the sample, whenpassing the sample through the porous membrane, the labeled antibodyrecognizes the analytic object to be measured and is bonded thereto. Thecomposite of the analytic object and the labeled antibody is flowed bycapillary phenomenon toward another porous membrane having the secondantibody (hereinafter, sometimes referred to as a trap antibody)immobilized thereon, for example, in the form of strips, and the complexof the analytic object and the labeled antibody is recognized, trappedand detected as a visible signal (in the form of strips in this case).In the case where the immunochromatography technique is applied tosaliva as a sample, however, the composite of a labeled antibody andmutans streptococci is trapped in the membrane retaining the labeledantibody but does not efficiently flow by capillary phenomenon towardthe porous membrane having the trap antibody immobilized therein tocause such a problem that the accuracy of the measurement is reduced.

SUMARY OF THE INVENTION

An object of the invention is to solve the problems associated with theconventional technique for identification and quantitative determinationof mutans streptococci, as one of cariogenic bacteria in human saliva,by immunochromatography utilizing an antigen-antibody reaction, and toprovide a pretreatment kit for saliva and a pretreatment method forsaliva in that aggregation caused by mucin and chain formation of mutansstreptococci in saliva can be removed in a simple operation, and acomplex of a labeled antibody and mutans streptococci effectively flowsout from a porous membrane retaining the labeled antibody.

As a result of earnest investigations made by the inventors for solvingthe problems, it has been found that the following effects can beobtained by treating with particular acid and alkali solutions tocomplete the present invention.

(1). Mucin and glucan in saliva are dissolved to act on an adventitia ofmutans streptococci to suppress aggregation.

(2) Upon using a particular surface active agent in addition thereto, aprotein in mutans streptococci is solubilized, whereby the mutansstreptococci is efficiently flowed through the porous membrane.

(3) Upon using a particular metallic salt, i.e., sodium chloride,potassium chloride, calcium chloride, magnesium chloride, magnesiumsulfate or manganese sulfate, in addition thereto, the complex of alabeled antibody and mutans streptococci can be efficiently flowed outfrom the membrane retaining the labeled antibody.

The present invention relates to, as one aspect, a pretreatment kit forsaliva containing

(A) an aqueous solution of sodium hydroxide having a concentration of0.01 to 10 mol/L,

(B) an aqueous solution of tartaric acid and/or citric acid having aconcentration of 0.01 to 3 mol/L, and

(C) a nonionic surface active agent and/or an amphoteric surface activeagent,

the component (C) being mixed with at least one of the components (A)and (B), or being provided separately from the components (A) and (B),and

at least one substance selected from the group consisting of sodiumchloride, potassium chloride, calcium chloride, magnesium chloride,magnesium sulfate and manganese sulfate being contained in at least oneof the components (A), (B) and (C) in an amount of 5 to 25% by weight.It is preferred in this aspect that (D) tris(hydroxymethyl)aminomethaneis mixed with at least one of the components (A), (B) and (C).

The present invention also relates to, as another aspect, a pretreatmentkit for saliva containing

(A) an aqueous solution of sodium hydroxide having a concentration of0.01 to 10 mol/L,

(B) an aqueous solution of tartaric acid and/or citric acid having aconcentration of 0.01 to 3 mol/L,

(C) a nonionic surface active agent and/or an amphoteric surface activeagent, and

(D) an aqueous solution containing tris(hydroxymethyl)aminomethane,

the component (C) being mixed with at least one of the components (A),(B) and (D), or being provided separately from the components (A), (B)and (D), and

at least one substance selected from the group consisting of sodiumchloride, potassium chloride, calcium chloride, magnesium chloride,magnesium sulfate and manganese sulfate being contained in at least oneof the components (A), (B), (C) and (D) in an amount of 5 to 25% byweight.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is preferred in the pretreatment kit for saliva of the presentinvention that the nonionic surface active agent as the component (C) isone kind or a mixture of two or more kinds selected from the groupconsisting of polyethylene glycol monooctylphenyl ether,n-octyl-β-D-glucoside, n-heptyl-β-D-thioglucoside,n-octyl-β-D-thioglucoside, nonylphenoxy polyethoxyethanol andpolyoxyethylene sorbitan monooleate, and the amphoteric surface activeagent as the component (C) is one kind or a mixture of two or more kindsselected from the group consisting of CHAPS(3-((3-cholamide-propyl)-dimethylammonio)-1-propanesulfonate) and CHAPSO(3-((3-cholamide-propyl)-dimethylammonio)-1-hydroxypropanesulfonate).

A pretreatment method for saliva in identification and quantitativedetermination of mutans streptococci by immunochromatography using thepretreatment kit for saliva of the present invention contains, as oneaspect, steps of mixing at least one substance selected from the groupconsisting of sodium chloride, potassium chloride, calcium chloride,magnesium chloride, magnesium sulfate and manganese sulfate with atleast one of (A) an aqueous solution of sodium hydroxide having aconcentration of 0.01 to 10 mol/L, (B) an aqueous solution of tartaricacid and/or citric acid having a concentration of 0.01 to 3 mol/L, and(C) a nonionic surface active agent and/or an amphoteric surface activeagent, in an amount of 5 to 25% by weight; and mixing the components(A), (B) and (C) by dropping in an arbitrary order (hereinafter,referred to as a first method).

The pretreatment method for saliva in identification and quantitativedetermination of mutans streptococci by immunochromatography alsocontains, as another aspect, steps of mixing at least one substanceselected from the group consisting of sodium chloride, potassiumchloride, calcium chloride, magnesium chloride, magnesium sulfate andmanganese sulfate with at least one of (A) an aqueous solution of sodiumhydroxide having a concentration of 0.01 to 10 mol/L and (B) an aqueoussolution of tartaric acid and/or citric acid having a concentration of0.01 to 3 mol/L, in an amount of 5 to 25% by weight; mixing (C) anonionic surface active agent and/or an amphoteric surface active agentin at least one of the components (A) and (B); and mixing the components(A) and (B) by dropping in an arbitrary order (hereinafter, referred toas a second method).

The pretreatment method for saliva in identification and quantitativedetermination of mutans streptococci by immunochromatography alsocontains, as another aspect, steps of mixing at least one substanceselected from the group consisting of sodium chloride, potassiumchloride, calcium chloride, magnesium chloride, magnesium sulfate andmanganese sulfate with at least one of (A) an aqueous solution of sodiumhydroxide having a concentration of 0.01 to 10 mol/L, (B) an aqueoussolution of tartaric acid and/or citric acid having a concentration of0.01 to 3 mol/L, (C) a nonionic surface active agent and/or anamphoteric surface active agent, and (D)tris(hydroxymethyl)aminomethane, in an amount of 5 to 25% by weight; andmixing the components (A), (B), (C) and (D) by dropping in such an orderthat the component (A) is in contact with the component (B) in thepresence of the component (D) (hereinafter, referred to as a thirdmethod).

It is preferred in the first method that (D)tris(hydroxymethyl)aminomethane is mixed in at least one of thecomponents (A), (B) and (C), and the components (A), (B) and (C) aremixed by dropping in such an order that the component (A) is in contactwith the component (B) in the presence of the component (D). It ispreferred in the second method that (D) tris(hydroxymethyl)aminomethaneis mixed in at least one of the components (A) and (B), and thecomponents (A) and (B) are mixed by dropping in an arbitrary order. Itis preferred in the third method that the component (A), (B) and (D), atleast one of which is mixed with the component (C), are mixed bydropping in such an order that the component (A) is in contact with thecomponent (B) in the presence of the component (D).

The aqueous solution of sodium hydroxide having a concentration of 0.01to 10 mol/L as the component (A) used in the pretreatment kit for salivaand the pretreatment method for saliva according to the presentinvention exerts a function to act on mucin in saliva and glucan presenton an adventitia of mutans streptococci to suppress aggregation ofmutans streptococci, so as to facilitate migration of the mutansstreptococci as an antigen in the porous membrane. The use of sodiumhydroxide as an alkali solution is essential, but sodium carbonate,disodium hydrogen phosphate and the like are not suitable, and theexamination of mutans streptococci cannot be carried out with otheralkali solutions than sodium hydroxide. It is supposed that this isbecause an alkali treatment other than sodium hydroxide may impair thestructure of the antigen of the mutans streptococci. The concentrationof sodium hydroxide in the aqueous solution is necessarily 0.01 to 10mol/L, and a concentration less than 0.01 mol/L cannot providesufficient effect, whereas that exceeding 10 mol/L breaks the antigen ofthe mutans streptococci to deteriorate the detection accuracy.

The aqueous solution of tartaric acid and/or citric acid having aconcentration of 0.01 to 3 mol/L as the component (B) used in thepretreatment kit for saliva and the pretreatment method for salivaaccording to the present invention exerts a function to suppress thechain formation of the mutans streptococci, so as to facilitatemigration of the mutans streptococci as an antigen in the porousmembrane. The use of tartaric acid and/or citric acid as an acid isessential, but other acids, such as hydrochloric acid, sulfuric acid,nitric acid, acetic acid, lactic acid and maleic acid, are not suitable,and the objective accuracy in examination cannot be obtained even whenthe other acids are used in combination with sodium hydroxide. It issupposed that this is because the other acids than tartaric acid andcitric acid may impair the structure of the antigen of the mutansstreptococci. The concentration of tartaric acid and/or citric acid inthe aqueous solution is necessarily 0.01 to 3 mol/L, and a concentrationless than 0.01 mol/L cannot provide sufficient effect, whereas thatexceeding 3 mol/L is not suitable because the solubility of tartaricacid and/or citric acid comes to the limit to form precipitation.

The nonionic surface active agent and/or the amphoteric surface activeagent as the component (C) used in the pretreatment kit for saliva andthe pretreatment method for saliva according to the present inventionexerts a function to solubilize a protein present on the surface ofmutans streptococci, so as to facilitate efficient flow of the mutansstreptococci through the porous membrane. An ionic surface active agenthas been often used in immunochromatography for facilitating smoothmigration of a sample solution or an antigen solution within anexamination apparatus. However, the surface active agent used in thepretreatment kit for saliva and the pretreatment method for saliva usedfor identification and quantitative determination of mutans streptococciaccording to the present invention is necessarily a nonionic surfaceactive agent and/or an amphoteric surface active agent as a result ofexperimentation, but detection of an antigen with an antibody cannot beattained with an anionic surface active agent, such as sodium laurylsulfate and sodium dodecylbenzenesulfonate.

The surface active agent used the present invention may be any nonionicsurface active agent and/or amphoteric surface active agent withoutparticular limitation, and any of those that are generally used as asolubilizing agent for a membrane protein can be used. The detectionsensitivity of mutans streptococci varies depending on the species ofthe nonionic surface active agent and/or the amphoteric surface activeagent used, and it is preferred to use one kind or a mixture of two ormore kinds selected from the group consisting of polyethylene glycolmonooctylphenyl ether, n-octyl-β-D-glucoside, n-heptyl-β-D-thioglucosideand n-octyl-β-D-thioglucoside as the nonionic surface active agent, andone kind or a mixture of two or more kinds selected from the groupconsisting of CHAPS(3-((3-cholamide-propyl)-dimethylammonio)-1-propanesulfonate) and CHAPSO(3-((3-cholamide-propyl)-dimethylammonio)-1-hydroxypropanesulfonate) asamphoteric surface active agent, from the standpoint of detectionsensitivity.

In the pretreatment kit for saliva and the pretreatment method forsaliva according to the present invention, the nonionic surface activeagent and/or the amphoteric surface active agent (C) is preferably usedto provide a concentration after treatment saliva of 0.05 to 90% byweight. In the case where the concentration is less than 0.05% byweight, it is not preferred since there is such a tendency that thedetection sensitivity by the antigen-antibody reaction is lowered, andin the case where it exceeds 90% by weight, it is also not preferredsince the detection sensitivity by the antigen-antibody reaction isliable to be lowered.

In the pretreatment kit for saliva and the pretreatment method forsaliva according to the present invention, the nonionic surface activeagent and/or the amphoteric surface active agent (C) may be providedseparately from the aqueous solution of sodium hydroxide having aconcentration of 0.01 to 10 mol/L (A) and the aqueous solution oftartaric acid and/or citric acid having a concentration of 0.01 to 3mol/L (B), and in this case, it may be in the form of an aqueoussolution. It may also be provided as a mixture with one or both of theaqueous solution of sodium hydroxide having a concentration of 0.01 to10 mol/L (A) and the aqueous solution of tartaric acid and/or citricacid having a concentration of 0.01 to 3 mol/L (B), and in this case,attentions are necessarily paid on decomposition property due to acidsand alkalis.

In the pretreatment kit for saliva and the pretreatment method forsaliva according to the present invention, at least one substanceselected from the group consisting of sodium chloride, potassiumchloride, calcium chloride, magnesium chloride, magnesium sulfate andmanganese sulfate is contained in at least one of the aqueous solutionof sodium hydroxide having a concentration of 0.01 to 10 mol/L (A), theaqueous solution of tartaric acid and/or citric acid having aconcentration of 0.01 to 3 mol/L (B) and the nonionic surface activeagent and/or the amphoteric surface active agent (C), in an amount of 5to 25% by weight in order to have the complex of the labeled antibodyand mutans streptococci flown out from the membrane retaining thelabeled antibody efficiently. The at least one substance selected fromthe group consisting of sodium chloride, potassium chloride, calciumchloride, magnesium chloride, magnesium sulfate and manganese sulfateexerts a function to aggregate various kinds of proteins present insaliva by salting out and counteract the mutual action between thelabeled antibody and the membrane retaining the same, so as to provideby the function an effect of facilitating efficient flow of the complexof the labeled antibody and the mutans streptococci from the membraneretaining the labeled antibody.

The at least one substance selected from the group consisting of sodiumchloride, potassium chloride, calcium chloride, magnesium chloride,magnesium sulfate and manganese sulfate is necessarily contained in atleast one of the aqueous solution of sodium hydroxide having aconcentration of 0.01 to 10 mol/L (A), the aqueous solution of tartaricacid and/or citric acid having a concentration of 0.01 to 3 mol/L (B)and the nonionic surface active agent and/or the amphoteric surfaceactive agent (C), in an amount of 5 to 25% by weight. In the case wherethe amount is less than 5% by weight, the effect cannot be sufficientlyobtained, and the complex of the labeled antibody and the mutansstreptococci cannot be efficiently flowed out from the membraneretaining the labeled antibody. In the case where it exceeds 25% byweight, on the other hand, the detection accuracy is ratherdeteriorated.

In the pretreatment kit for saliva and the pretreatment method forsaliva according to the present invention, a buffer agent is preferablyused since a neutralizing reaction occurs between the aqueous solutionof sodium hydroxide having a concentration of 0.01 to 10 mol/L (A) andthe aqueous solution of tartaric acid and/or citric acid having aconcentration of 0.01 to 3 mol/L (B), and in order to obtain a bufferingaction efficiently in the neutralizing reaction,tris(hydroxymethyl)aminomethane is preferably used as the buffer agent.At this time, however, it has been confirmed that no sufficientbuffering action can be obtained with other buffer agents, such as acombination of sodium hydrogencarbonate and sodium carbonate, and acombination of citric acid and sodium citrate. Thetris(hydroxymethyl)aminomethane may be mixed with one of the components(A), (B) and (C), or in alternative, it may be provided separately fromthem, preferably in the form of an aqueous solution. In the pretreatmentkit for saliva according to the present invention, it is necessary thatthe aqueous solution of sodium hydroxide having a concentration of 0.01to 10 mol/L (A) and the aqueous solution of tartaric acid and/or citricacid having a concentration of 0.01 to 3 mol/L (B) are providedseparately from each other because they cause neutralization.

In the first method according to the present invention, uponidentification and quantitative determination of mutans streptococci byimmunochromatography, at least one substance selected from the groupconsisting of sodium chloride, potassium chloride, calcium chloride,magnesium chloride, magnesium sulfate and manganese sulfate is mixedwith at least one of the components (A), (B) and (C), in an amount of 5to 25% by weight, and then the aqueous solution of sodium hydroxidehaving a concentration of 0.01 to 10 mol/L (A), the aqueous solution oftartaric acid and/or citric acid having a concentration of 0.01 to 3mol/L (B) and the nonionic surface active agent and/or the amphotericsurface active agent (C) are mixed by dropping in an arbitrary order. Inorder to simplify the operation in the second method according to thepresent invention, the nonionic surface active agent and/or theamphoteric surface active agent (C) is mixed with at least one of theaqueous solution of sodium hydroxide having a concentration of 0.01 to10 mol/L (A) and the aqueous solution of tartaric acid and/or citricacid having a concentration of 0.01 to 3 mol/L (B), and then the aqueoussolution of sodium hydroxide having a concentration of 0.01 to 10 mol/L(A) and the aqueous solution of tartaric acid and/or citric acid havinga concentration of 0.01 to 3 mol/L (B) are mixed by dropping in anarbitrary order.

In the pretreatment method for saliva according to the presentinvention, a buffer agent is preferably used since a neutralizingreaction occurs between the aqueous solution of sodium hydroxide havinga concentration of 0.01 to 10 mol/L (A) and the aqueous solution oftartaric acid and/or citric acid having a concentration of 0.01 to 3mol/L (B), and in order to obtain a buffering action efficiently in theneutralizing reaction, tris(hydroxymethyl)aminomethane (D) is preferablyused. While the treatments with the components (A), (B) and (C) may becarried out in an arbitrary order since they function independently fromeach other, it is necessary in the case where thetris(hydroxymethyl)aminomethane (D) is contained in the pretreatment kitfor saliva that the components (A) and (B) are in contact with eachother in the presence of the tris(hydroxymethyl)aminomethane (D) toobtain a buffering action.

In addition to the first and second methods, in the third methodaccording to the present invention, upon identification and quantitativedetermination of mutans streptococci by immunochromatography, at leastone substance selected from the group consisting of sodium chloride,potassium chloride, calcium chloride, magnesium chloride, magnesiumsulfate and manganese sulfate is mixed with at least one of the aqueoussolution of sodium hydroxide having a concentration of 0.01 to 10 mol/L(A), the aqueous solution of tartaric acid and/or citric acid having aconcentration of 0.01 to 3 mol/L (B), the nonionic surface active agentand/or an amphoteric surface active agent (C), and thetris(hydroxymethyl)aminomethane (D), in an amount of 5 to 25% by weight,and then the components (A), (B), (C) and (D) are mixed by dropping insuch an order that the component (A) is in contact with the component(B) in the presence of the component (D). It is preferred in this casethat the component (A), (B) and (D), at least one of which is mixed withthe component (C), are mixed by dropping in such an order that thecomponent (A) is in contact with the component (B) in the presence ofthe component (D).

In the pretreatment method for saliva according to the presentinvention, the treatment is preferably carried out in such a manner thatthe pH value of saliva after the treatment is in a range of 5 to 9 sincethe antigen-antibody reaction is carried out within the pH range. Whilethe range varies depending on the antibody used, there is such atendency that the reliability of the measurement result is lowered whenthe pH is outside the range since the antibody and the antigen arereleased from each other, and they exert nonspecific affinity.

The sample of saliva having been treated by the pretreatment kit forsaliva and the pretreatment method for saliva according to the presentinvention can be subjected to identification and quantitativedetermination of mutans streptococci by an antigen-antibody reactionusing the conventional immunochromatography technique. The antibody canbe obtained by an ordinarily employed way. For example, it may beobtained by the establishment of hybridoma by cell fusion according toKohler and Milstein (Kohler G, C. Milstein, Continuous cultures of fusedcells secreting antibody of predefined specificity, Nature, vol. 256, p.495-497 (1975)), or may be those simply purified from a serum of ananimal having been immunized with the antigen.

EXAMPLE

The present invention will be described in detail below with referenceto examples, but the present invention is not construed as being limitedthereto. Unless otherwise specified, the operations were carried out atroom temperature, and the pH values are those at a temperature at 20 to25° C.

(1) Preparation of Reagent and Test Device

1. Production of Antibodies

Streptococci mutans (ATCC 25175 strain) and Streptococci sobrinus (ATCC33478 strain) were cultured by using a BHI culture medium (Brain HeartInfusion culture medium, produced by Difco Laboratories) at 37° C.overnight, respectively. After collecting bacteria cells from theculture solution by centrifugation, they were washed twice with PBS (10mmol phosphate buffered saline), and the growth was terminated with aformaldehyde aqueous solution. Mice were directly immunized with thedispersion of the bacteria, and the following two kinds each of purifiedantibodies were obtained by the establishment of hybridoma using cellfusion by Kohler and Milstein. SM1 antibody: Antibody againstStreptococci mutans SM2 antibody: Antibody against Streptococci mutansSS1 antibody: Antibody against Streptococci sobrinus SS2 antibody:Antibody against Streptococci sobrinus

2. Gold Colloid Labeling of Antibody

The SM2 antibody and the SS2 antibody were labelled with a gold colloidhaving a particle diameter of 40 nm. A commercially available goldcolloid (produced by British Biocell International) was used, anddiluted with PBS having 1% of bovine serum albumin (BSA, a trade name,produced by Sigma-Aldrich Corp.) and 1% of a nonionic surface activeagent (Tween 20, a trade name, produced by Sigma-Aldrich Corp.) addedthereto to an antibody concentration of 0.1 μg/mL. The antibodysolutions labelled with the gold colloid are referred to as SM2 labelledantibody and SS2 labelled antibody, respectively.

3. Production of Antibody-Immobilized Porous Film Strip

A nitrocellulose membrane lined with a plastic film (SXHF, a trade name,produced by Millipore Corp. Japan) was used as a porous film. Themembrane was cut into a rectangle of 5 mm×40 mm. The SM1 antibody or theSS1 antibody was diluted with a 50 mmol phosphate buffer solutioncontaining 1% of bovine serum albumin to a concentration of 1 mg/mL, andthe diluted solution of the antibody was applied on a central part ofthe nitrocellulose membrane thus cut in the direction perpendicular tothe longitudinal direction of the membrane with a micropipet in anamount of about 1 μL/cm. The immobilized antibodies are sometimesreferred to as SM1 trap antibody and SS1 trap antibody. Filter paper of15 mm square as a water absorbing body was fixed on one end of themembrane with a clip to contact closely with each other. A polypropylenematrix (Quick Release Conjugate Pad, a trade name, produced by MilliporeCorp. Japan) of 5 mm by 20 mm as a porus membrane retaining the labelledantibody (hereinafter, referred to as a labelled antibody retention) wasfixed on the other end of the membrane opposite to the water absorbingbody with a clip, on which 30 μL of the SM2 labelled antibody solutionor the SS2 labelled antibody solution was dropped. The device was driedat 37° C. for 2 hours and stored in a desiccator until the time of use.

(2) Quantitative Determination of Mutans Streptococci

The results of quantitative determination of mutans streptococci insaliva by the immunochromatography using the foregoing reagents anddevice was compared with the number of mutans streptococci in saliva bythe conventional method of calculation from the number of colonies.

1. Examination Method by Immunochromatography

The reactivity between the mutans streptococci and the antibodyimmobilized on the antibody-immobilized porous membrane was detected bythe following principle. Upon passing saliva through the labelledantibody retention of the antibody-immobilized porous membrane strip,the labelled antibody was bonded to the mutans streptococci in saliva tocolor in red. The complex of the mutans streptococci and the labelledantibody migrated in the antibody-immobilized porous membrane strip, andit was then trapped by the antibody (trap antibody) immobilized, forexample, in the form of strips, in the antibody-immobilized porousmembrane strip and thus confirmed as a strip-shape mark.

2. Number of Mutans Streptococci by Conventional Calculation from Numberof Colonies

Streptococci mutans, Streptococci sobrinus and foreign bacteria formedcolonies on an MSB culture medium. In order that the number of coloniesof them were accurately measured, after measuring the number of coloniesformed on the MSB culture medium, a part of the colonies was collectedand crushed, and the PCR (polymerase chain reaction) is carried out witha synthetic DNA primer having a sequence that is specific inStreptococci mutans or Streptococci sobrinus. The thus amplified genefragments were subjected to electrophoresis with agarose gel to confirmthe strain.

Example 1

Such an antibody-immobilized porous film strip was used that has SM1antibody immobilized at the central part thereof and SM2 labelledantibody contained in a labelled antibody retention at one end thereof.The examination was carried out in the following manner.

An examination subject was made manducate gum for collecting saliva for5 minutes to collect saliva in a test tube. A part of the saliva thuscollected was diluted with PBS and applied on an MSB culture medium.After anaerobically culturing at 37° C. for 2 or 3 days, the number ofcolonies was measured. After the measurement, a part of the colonies wascollected. After crushing the bacteria cells, a PCR (polymerase chainreaction) was carried out with a synthetic DNA primer having a sequencethat is specific in Streptococci mutans or Streptococci sobrinus, andthe amplified gene fragments were subjected to electrophoresis withagarose gel to discriminate the strains. The number of bacteria cells(CFU/mL) was calculated from the number of colonies that were confirmedto be those of Streptococci mutans as a result of the discrimination.

A solution (AD solution) was prepared by adding 23.3% by weight ofsodium chloride to a solution containing 1.0 mol/L oftris(hydroxymethyl)aminomethane (component (D)) containing 1.0 mol/L ofsodium hydroxide (component (A)). A solution (BCD solution) was preparedby adding 5% by weight of polyethylene glycol monooctylphenyl ether(produced by Wako Pure Chemical Industries, Ltd.) as a nonionic surfaceactive agent (component (C)) to a solution containing 1.0 mol/L oftris(hydroxymethyl)aminomethane (component (D)) containing 0.5 mol/L ofcitric acid (component (B)).

50 μL of the AD solution was added to 250 μL of the saliva, followed bystirring, and then 100 μL of the BCD solution was added thereto,followed by stirring. 100 μL out of the saliva thus treated was droppedon the strip on the side of the labelled antibody retention. Thesolution thus treated had pH of 7.2. The concentration of the component(C) in the solution was 1.25% by weight. After 15 minutes from thedropping, the reaction with the trap antibody was confirmed, and theamount of the complex of the SM2 labelled antibody and Streptococcimutans remaining in the labelled antibody retention was visuallyobserved.

The examination described in the foregoing was carried out for fivesubjects a to e. The results thus obtained are shown in Table 1 below.The reactivity with the antibody was evaluated for the four grades,i.e., a red strip could be clearly recognized (++), a red strip could berecognized (+), a red strip could be slightly recognized (±), and nostrip was recognized (−). The amount of the complex of the SM2 labelledantibody and Streptococci mutans remaining in the labelled antibodyretention (hereinafter, simply referred to as a remaining antibodyamount) was visually observed and evaluated for the three grades, i.e.,a large amount of the remaining antibody could be confirmed as red colorin the labelled antibody retention (+), the remaining antibody could beslightly confirmed (±), and none of them was confirmed (−). The unit,CFU/mL, used in the table means the number of colonies of the bacteriumper 1 mL of the saliva. TABLE 1 Number of colonies of Streptococcimutans Reaction Amount of remaining on MSB culture medium with antibodyin labelled Subject (CFU/mL) antibody antibody retention a 1.2 × 10⁴ − −b 2.1 × 10⁵ ± − c 4.5 × 10⁵ + − d 7.3 × 10⁵ + ± e 9.2 × 10⁵ + + ±

Example 2

The examination was carried out in the following manner by using anantibody-immobilized porous film strip that was the same as in Example 1except that SS1 trap antibody was used instead of the SM1 trap antibody,and SS2 labelled antibody was used instead of the SM2 labelled antibody.

A solution (A solution) was prepared by adding 25.0% by weight of sodiumchloride to a solution containing 1.0 mol/L of sodium hydroxide(component (A)). A solution (BD solution) was prepared by adding 0.5mol/L of citric acid (component (B)) to absolution containing 1.0 mol/Lof tris(hydroxymethyl)aminomethane (component (D)). An aqueous solution(C solution) containing 5% by weight of polyethylene glycolmonooctylphenyl ether (produced by Wako Pure Chemical Industries, Ltd.)as a nonionic surface active agent (component (C)) was prepared.

80 μL of the BD solution was added to 250 μL of the saliva, followed bystirring, 50 μL the C solution was then added thereto, followed bystirring, and 40 μL of the A solution was finally added thereto,followed by stirring. 100 μL out of the saliva thus treated was droppedon the strip on the side of the labelled antibody retention. Thesolution thus treated had pH of 6.6. The concentration of the component(C) in the solution was 0.60% by weight. After 15 minutes from thedropping, the reaction with the trap antibody was confirmed, and theamount of the complex of the SS2 labelled antibody and Streptococcisobrinus remaining in the labelled antibody retention was visuallyobserved.

The examination described in the foregoing was carried out for fivesubjects a to e, and evaluations were carried out in the same manner asin Example 1. The results thus obtained are shown in Table 2 below.TABLE 2 Number of colonies of Streptococci sobrinus Reaction Amount ofremaining on MSB culture medium with antibody in labelled Subject(CFU/mL) antibody antibody retention a 1.4 × 10⁴ − ± b 4.0 × 10⁵ + − c4.4 × 10⁵ + ± d 9.0 × 10⁵ + + − e 9.3 × 10⁵ + + −

Example 3

The examination was carried out in the following manner by using anantibody-immobilized porous film strip using the SM1 trap antibody andthe SM2 labelled antibody that was the same as in Example 1.

A solution (AC solution) was prepared by adding 1.0 mol/L of sodiumhydroxide (component (A)) to an aqueous solution containing 5% by weightof polyethylene glycol monooctylphenyl ether (produced by Wako PureChemical Industries, Ltd.) as a nonionic surface active agent (component(C)). A solution (BD solution) was prepared by adding 12.0% by weight ofsodium chloride to a solution containing 1.0 mol/L oftris(hydroxymethyl)aminomethane (component (D)) containing 0.5 mol/L ofcitric acid (component (B)).

75 μL of the AC solution was added to 250 μL of the saliva, followed bystirring, and 75 μL of the BD solution was then added thereto, followedby stirring. 100 μL out of the saliva thus treated was dropped on thestrip on the side of the labelled antibody retention. The solution thustreated had pH of 7.5. The concentration of the component (C) in thesolution was 0.94% by weight. After 15 minutes from the dropping, thereaction with the trap antibody was confirmed, and the amount of thecomplex of the SM2 labelled antibody and Streptococci mutans remainingin the labelled antibody retention was visually observed.

The examination described in the foregoing was carried out for threesubjects a to c, and evaluations were carried out in the same manner asin Example 1. The results thus obtained are shown in Table 3 below.TABLE 3 Number of colonies of Streptococci mutans Reaction Amount ofremaining on MSB culture medium with antibody in labelled Subject(CFU/mL) antibody antibody retention a 1.2 × 10⁴ − − b 4.3 × 10⁵ + − c8.4 × 10⁵ + + −

Example 4

The examination was carried out in the following manner by using anantibody-immobilized porous film strip using the SS1 trap antibody andthe SS2 labelled antibody that was the same as in Example 2.

An aqueous solution (A solution) containing 4.0 mol/L of sodiumhydroxide (component (A)) was prepared. An aqueous solution (B solution)containing 1.0 mol/L of tartaric acid (component (B)) was prepared. Anaqueous solution (C solution) containing 5% by weight of polyethyleneglycol monooctylphenyl ether (produced by Wako Pure Chemical Industries,Ltd.) as a nonionic surface active agent (component (C)) was prepared. Asolution (D solution) was prepared by adding 12.0% by weight of sodiumchloride to a solution containing 1.0 mol/L oftris(hydroxymethyl)aminomethane (component (D)).

40 μL of the C solution was added to 250 μL of the saliva, followed bystirring, 40 μL of the B solution was then added thereto, followed bystirring, 40 μL of the D solution was further added thereto, followed bystirring, and 40 μL of the A solution was finally added thereto,followed by stirring. 100 μL out of the saliva thus treated was droppedon the strip on the side of the labelled antibody retention. Thesolution thus treated had pH of 8.0. The concentration of the component(C) in the solution was 0.49% by weight. After 15 minutes from thedropping, the reaction with the trap antibody was confirmed, and theamount of the complex of the SS2 labelled antibody and Streptococcisobrinus remaining in the labelled antibody retention was visuallyobserved.

The examination described in the foregoing was carried out for threesubjects a to c, and evaluations were carried out in the same manner asin Example 1. The results thus obtained are shown in Table 4 below.TABLE 4 Number of colonies of Streptococci sobrinus Reaction Amount ofremaining on MSB culture medium with antibody in labelled Subject(CFU/mL) antibody antibody retention a 1.0 × 10⁴ − − b 3.9 × 10⁵ + ± c7.7 × 10⁵ + + −

Example 5

The examination was carried out in the following manner by using anantibody-immobilized porous film strip using the SM1 trap antibody andthe SM2 labelled antibody that was the same as in Example 1.

A solution (AC solution) was prepared by adding 12.0% by weight ofsodium chloride to an aqueous solution containing 5% by weight ofpolyethylene glycol monooctylphenyl ether (produced by Wako PureChemical Industries, Ltd.) as a nonionic surface active agent (component(C)) containing 1.0 mol/L of sodium hydroxide (component (A)). Anaqueous solution (B solution) containing 0.5 mol/L of tartaric acid(component (B)) was prepared. A solution (D solution) was prepared byadding 12.0% by weight of sodium chloride to a solution containing 1.0mol/L of tris(hydroxymethyl)aminomethane (component (D)).

75 μL of the AC solution was added to 250 μL of the saliva, followed bystirring, 75 μL of the B solution was then added thereto, followed bystirring, and 50 μL of the D solution was finally added thereto,followed by stirring. 100 μL out of the saliva thus treated was droppedon the strip on the side of the labelled antibody retention. Thesolution thus treated had pH of 7.2. The concentration of the component(C) in the solution was 0.83% by weight. After 15 minutes from thedropping, the reaction with the trap antibody was confirmed, and theamount of the complex of the SM2 labelled antibody and Streptococcimutans remaining in the labelled antibody retention was visuallyobserved.

The examination described in the foregoing was carried out for threesubjects a to c, and evaluations were carried out in the same manner asin Example 1. The results thus obtained are shown in Table 5 below.TABLE 5 Number of colonies of Streptococci mutans Reaction Amount ofremaining on MSB culture medium with antibody in labelled Subject(CFU/mL) antibody antibody renention a 1.2 × 10⁴ − ± b 4.3 × 10⁵ + − c8.4 × 10⁵ + + −

Example 6

The examination was carried out in the following manner by using anantibody-immobilized porous film strip using the SM1 trap antibody andthe SM2 labelled antibody that was the same as in Example 1.

A solution (AD solution) was prepared by adding 12.0% by weight ofsodium chloride to a solution containing 1.0 mol/L oftris(hydroxymethyl)aminomethane (component (D)) containing 3.0 mol/L ofsodium hydroxide (component (A)). A solution (BD solution) was preparedby adding 1.0 mol/L of tartaric acid and 0.5 mol/L of citric acid(component (B)) to a solution containing 1.0 mol/L oftris(hydroxymethyl)aminomethane (component (D)). A solution (C solution)was prepared by adding 2 mol/L of sodium chloride to an aqueous solutioncontaining 5% by weight of polyethylene glycol monooctylphenyl ether(produced by Wako Pure Chemical Industries, Ltd.) as a nonionic surfaceactive agent (component (C)).

40 μL of the BD solution was added to 250 μL of the saliva, followed bystirring, 40 μL of the A solution was then added thereto, followed bystirring, and 40 μL of the C solution was finally added thereto,followed by stirring. 100 μL out of the saliva thus treated was droppedon the strip on the side of the labelled antibody retention. Thesolution thus treated had pH of 6.9. The concentration of the component(C) in the solution was 0.54% by weight. After 15 minutes from thedropping, the reaction with the trap antibody was confirmed, and theamount of the complex of the SM2 labelled antibody and Streptococcimutans remaining in the labelled antibody retention was visuallyobserved.

The examination described in the foregoing was carried out for threesubjects a to c, and evaluations were carried out in the same manner asin Example 1. The results thus obtained are shown in Table 6 below.TABLE 6 Number of colonies of Streptococci mutans Reaction Amount ofremaining on MSB culture medium with antibody in labelled Subject(CFU/mL) antibody antibody retention a 1.1 × 10⁴ − − b 3.9 × 10⁵ + − c7.7 × 10⁵ + + −

Example 7

The examination was carried out in the following manner by using anantibody-immobilized porous film strip using the SM1 trap antibody andthe SM2 labelled antibody that was the same as in Example 1.

A solution (A solution) was prepared by adding 25.0% by weight of sodiumchloride to an aqueous solution containing 1.0 mol/L of sodium hydroxide(component (A)). A solution (BC solution) was prepared by adding 5% byweight of polyethylene glycol monooctylphenyl ether (produced by WakoPure Chemical Industries, Ltd.) as a nonionic surface active agent(component (C)) to an aqueous solution containing 0.5 mol/L of citricacid (component (B)). A buffer solution (D solution) containing 2 mol/Lof tris(hydroxymethyl)aminomethane (component (D)) was prepared.

40 μL of the D solution was added to 250 μL of the saliva, followed bystirring, 40 μL of the A solution was then added thereto, followed bystirring, and 70 μL of the BC solution was finally added thereto,followed by stirring. 100 μL out of the saliva thus treated was droppedon the strip on the side of the labelled antibody retention. Thesolution thus treated had pH of 6.9. The concentration of the component(C) in the solution was 0.88% by weight. After 15 minutes from thedropping, the reaction with the trap antibody was confirmed, and theamount of the complex of the SM2 labelled antibody and Streptococcimutans remaining in the labelled antibody retention was visuallyobserved.

The examination described in the foregoing was carried out for fivesubjects a to e, and evaluations were carried out in the same manner asin Example 1. The results thus obtained are shown in Table 7 below.TABLE 7 Number of colonies of Streptococci mutans Reaction Amount ofremaining on MSB culture medium with antibody in labelled Subject(CFU/mL) antibody antibody retention a 3.8 × 10⁴ − − b 1.6 × 10⁵ − − c4.7 × 10⁵ ± ± d 7.2 × 10⁵ + ± e 9.2 × 10⁵ + + −

Comparative Example 1

The same examination as in Example 1 was carried out for five subjects ato e except that the AD solution containing no sodium chloride was used,and evaluations were carried out in the same manner as in Example 1. Theresults thus obtained are shown in Table 8 below. TABLE 8 Number ofcolonies of Streptococci mutans Reaction Amount of remaining on MSBculture medium with antibody in labelled Subject (CFU/mL) antibodyantibody retention a 1.2 × 10⁴ − + b 2.1 × 10⁵ − + c 4.5 × 10⁵ ± + d 7.3× 10⁵ + + e 9.2 × 10⁵ + + +

Comparative Example 2

The same examination as in Example 4 was carried out for three subjectsa to c except that the D solution containing no sodium chloride wasused, and evaluations were carried out in the same manner as inExample 1. The results thus obtained are shown in Table 9 below. TABLE 9Number of colonies of Streptococci mutans Reaction Amount of remainingon MSB culture medium with antibody in labelled Subject (CFU/mL)antibody antibody retention a 1.0 × 10⁴ − + b 3.9 × 10⁵ ± + c 7.7 × 10⁵++ +

It was confirmed from the foregoing results that the pretreatment methodfor saliva by using the pretreatment kit for saliva according to thepresent invention could remove aggregation caused by mucin and chainformation of mutans streptococci in saliva, and since the amount of thecomplex of the labelled antibody and the mutans streptococci remainingin the labelled antibody retention was small, the effect of preventingsuch a phenomenon that the complex of the labelled antibody and themutans streptococci remained in the membrane retaining the labelledantibody was confirmed.

As having been described in detail, upon identification and quantitativedetermination of mutans streptococci, which is a type of cariogenicbacteria in human saliva, by immunochromatography utilizing anantigen-antibody reaction, the pretreatment kit for saliva and thepretreatment method for saliva according to the present invention canremove aggregation caused by mucin and chain formation of mutansstreptococci in saliva in a simple operation, and can efficiently flow acomplex of a labeled antibody and mutans streptococci from a porousmembrane retaining the labeled antibody, whereby identification andquantitative determination can be carried out with high accuracy.Therefore, it provides significant value by contributing to the field ofdentistry.

1-2. (canceled)
 3. A pretreatment kit for saliva comprising (A) anaqueous solution of sodium hydroxide having a concentration of 0.01 to10 mol/L, (B) an aqueous solution of tartaric acid and/or citric acidhaving a concentration of 0.01 to 3 mol/L, (C) a nonionic surface activeagent and/or an amphoteric surface active agent, and (D) an aqueoussolution containing tris(hydroxymethyl)aminomethane, the component (C)being mixed with at least one of the components (A), (B) and (D), orbeing provided separately from the components (A), (B) and (D), and atleast one substance selected from the group consisting of sodiumchloride, potassium chloride, calcium chloride, magnesium chloride,magnesium sulfate and manganese sulfate being contained in at least oneof the components (A), (B), (C) and (D) in an amount of 5 to 25% byweight. 4-5. (canceled)
 6. A pretreatment method for saliva inidentification and quantitative determination of streptococci mutans byimmunochromatography, which comprises: mixing at least one substanceselected from the group consisting of sodium chloride, potassiumchloride, calcium chloride, magnesium chloride, magnesium sulfate andmanganese sulfate with at least one of (A) an aqueous solution of sodiumhydroxide having a concentration of 0.01 to 10 mol/L, (B) an aqueoussolution of tartaric acid and/or citric acid having a concentration of0.01 to 3 mol/L, and (C) a nonionic surface active agent and/or anamphoteric surface active agent, in an amount of 5 to 25% by weight; andmixing the components (A), (B) and (C) by dropping in an arbitraryorder.
 7. A pretreatment method for saliva as claimed in claim 6,wherein (D) tris(hydroxymethyl)aminomethane is mixed in at least one ofthe components (A), (B) and (C), and the components (A), (B) and (C) aremixed by dropping in such an order that the component (A) is in contactwith the component (B) in the presence of the component (D).
 8. Apretreatment method for saliva in identification and quantitativedetermination of streptococci mutans by immunochromatography, whichcomprises: mixing at least one substance selected from the groupconsisting of sodium chloride, potassium chloride, calcium chloride,magnesium chloride, magnesium sulfate and manganese sulfate with atleast one of (A) an aqueous solution of sodium hydroxide having aconcentration of 0.01 to 10 mol/L and (B) an aqueous solution oftartaric acid and/or citric acid having a concentration of 0.01 to 3mol/L, in an amount of 5 to 25% by weight; and mixing (C) a nonionicsurface active agent and/or an amphoteric surface active agent in atleast one of the components (A) and (B); and mixing the components (A)and (B) by dropping in an arbitrary order.
 9. A pretreatment method forsaliva as claimed in claim 8, wherein (D)tris(hydroxymethyl)aminomethane is mixed in at least one of thecomponents (A) and (B), and the components (A) and (B) are mixed bydropping in an arbitrary order.
 10. A pretreatment method for saliva inidentification and quantitative determination of streptococci mutans byimmunochromatography, which comprises: mixing at least one substanceselected from the group consisting of sodium chloride, potassiumchloride, calcium chloride, magnesium chloride, magnesium sulfate andmanganese sulfate with at least one of (A) an aqueous solution of sodiumhydroxide having a concentration of 0.01 to 10 mol/L, (B) an aqueoussolution of tartaric acid and/or citric acid having a concentration of0.01 to 3 mol/L, (C) a nonionic surface active agent and/or anamphoteric surface active agent, and (D)tris(hydroxymethyl)aminomethane, in an amount of 5 to 25% by weight; andmixing the components (A), (B), (C) and (D) by dropping in such an orderthat the component (A) is in contact with the component (B) in thepresence of the component (D).
 11. A pretreatment method for saliva asclaimed in claim 10, wherein the component (A), (B) and (D), at leastone of which is mixed with the component (C), are mixed by dropping insuch an order that the component (A) is in contact with the component(B) in the presence of the component (D).